The aminoacyl-tRNA synthetases constitute a group of about twenty proteins which play a crucial role in translating the genetic code by catalyzing the reaction joining amino acids to their cognate tRNAs. Despite widespread common functionality, their structures are very diverse. Primary structures of almost all 20 bacterial and yeast synthetases have been ascertained. This research has led to the recognition of two major synthetase classes based upon both crystallographic and primary structure analysis. These findings have greatly advanced our understanding of the evolutionary pathways that led to the functional preservation of synthetases despite their remarkable structural diversity.
In contrast to the data from lower eukaryotes, far less structural information is available for synthetases from higher eukaryotes. The only nucleotide sequences of mammalian synthetases so far available are those of the glutaminyl-synthetase (Fett, R. et al. (1991) J. Biol. Chem. 266: 1448-1455), aspartyl-synthetase (Jacobo-Molina et al. (1989) J. Biol. Chem. 264: 16608-1612), threonyl-synthetase (Cruzen et al. (1991) J. Biol. Chem. 266: 9919-9923), valyl-synthetase (Hsieh et al. (1991)Biochem. J. 278: 809-816), tryptophanyl-synthetase (Garret et al. (1991) Biochemistry 30: 7809-7817) and histidyl-synthetase (Tsui et al. (1987) Nucleic. Acids. Res. 15: 3349-3367; Tsui et al. (1987) Gene 61: 349-361).
The histidyl-tRNA synthetase (HRS) has been particularly interesting since it is a frequent target of autoantibodies relating to human autoimmune diseases including myositis, polymyositis and dermatomyositis (Nishikai et al. (1980) Arthritis Rheum. 23: 881-888). Four other synthetases have also been found as targets of autoantibodies--threonyl-synthetase (Mathews et al. (1984) J. Exp. Med. 160: 42-434), alanyl-synthetase (Bunn et al. (1987) Mol. Biol. Med. 4: 21-36), glycyl-synthetase, and isoleucyl-synthetase (Targoff, I. N. (1990) J. Immunol. 144: 1737-1743). However, these synthetases were only rarely found to be an autoimmune target.
Patients having these autoantibodies form a distinct group clinically, and perhaps genetically (Love et al. (1991) Medicine (Baltimore) 70: 360-374). Recent studies on the mechanisms of anti-HRS induction and regulation suggest that the native human enzyme is the antigen which selects and sustains the immune response, preceding clinical illness (Miller et al. (1990) J. Clin. Invest. 85: 468-475; Miller et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87: 9933-9937).
Assays for detecting HRS in serum have been developed (Biswas et al. (1987), Journal of Immunological Methods 98: 243-248) and have proven very useful for detecting autoimmune diseases related to HRS in human serum. To produce these assays purified HRS protein was isolated from HeLa cells using high performance liquid chromatography (HPLC). The isolated HRS protein was then bound to an ELISA plate and incubated in the presence of human serum (Biswas et al. (1987), Journal of Immunological Methods 98: 243-248). Detectable binding of antibodies in the serum to the bound HRS protein indicated an autoimmune disease state.
HRS proteins have also been isolated from calves' liver (Targoff et al., J. Immunol., 138:2874-2882 (1987). However HRS proteins isolated from calves' liver or HeLa cells were very unstable, even when stored at -80.degree. C. For this reason, ELISA experiments using such isolated HRS proteins had to be performed rapidly following isolation. In addition, isolating HRS using HPLC is a time-consuming and difficult process.
The HRS gene from a SV40 transformed fibroblast cell line was cloned and expressed in COS 1 cells (Tsui et al. Nucl. Acid. Res. (1987) 8: 3349-3367). Three regions of extensive homology between the Human HRS and a hamster HRS clone were discovered. In addition, Ramsden et al. (Journal of Immunology (1989) 143: 2267-2272) has epitope mapped the expressed product of a cloned HRS gene. However, in these experiments the HRS cDNA was transiently transfected into COS 1 cells which already express an endogenous HRS. For this reason, background binding from expression of the endogenous enzyme made determining the amount of binding from the transfected clone difficult.
Although others have disclosed methods of isolating HRS from HELA cell extracts, these proteins were only weakly immunogenic in assays for autoimmune diseases. It would be very advantageous to provide a HRS which was much more sensitive to antibodies than the previously disclosed protein.